1. Field of the invention
This invention relates to an optical confocal scanning microscope.
2. Description of the Related Art
U.K. Patent Publication No. 2 184 321A discloses a confocal scanning optical microscope which is especially for the study of fluorescent or reflecting specimens. This instrument depends upon the focussing of light upon a single spot scanned over the specimen, which illuminated spot, after de-scanning, is imaged on a confocal aperture in front of a detector.
In the case where an image is to be formed of fluorescence from a specimen, the wavelength of the light directed on to the specimen is selected in such a way as to excite fluorescence. The emitted light is separated from the exciting light by a suitable beam splitter and is passed through wavelength-selective filters in such a way that the detector responds only to the light emitted by fluorescence. Instruments based on this design are commercially available. They contain a provision for subdividing the emitted light into beams of different wavelength ranges by a suitable beam splitter and filters. After this division, two dyes can be employed which emit different colours of fluorescence which can be distinguished at two detectors. Alternatively, a reflectance image can be obtained at the same time as a fluorescence image by the use of suitable beam-splitters, in accordance with accepted optical practice.
The prior art instruments work satisfactorily but all confocal scanning microscopes which rely on the use of a single scanning spot suffer from the defect that all the spectral selectivity of the system lies in the separation of the emitted or reflected beam into fractions of different wavelength. If there is considerable overlap between the fluorescent emission spectra of two dyes, they cannot be distinguished. For example, Bacallao et al comment in the Handbook of Confocal Microscopy, Plenum Press, 1990, that the commonly-used dyes fluorescein and rhodamine cannot be separated effectively in a system of this type. In order to achieve acceptable separation, it is necessary to vary the wavelength of excitation. This can be done by changing from one type of laser light to another, of spectrally different properties. First an image is obtained by operating the system with one type of excitation, and then a second image is obtained with a different type of exciting beam. This operation is slow and cumbersome.
Awamura, Ode and Yonezawa have described a microscope in which red, green and blue laser beams are scanned independently over the specimen, and the reflected beams are separated by dichroic filters and each executes a scanning motion over one of three separate linear CCD detector arrays. The description was published in the Proceedings of SPIE, The International Society for Optical Engineering (1987) Volume 765 pp 53-60. In principle, the system of Awamura et al might be used as a fluorescence microscope. It would then allow more than one type of dye to be excited in rapid succession during each line scan. However, in the case of two dyes with identical emission spectra, or a single ratiometric dye where the emission spectrum was to be monitored in a single waveband, the system of Awamura et al offers no advantage over that of White (U.K. Patent Application No. 2 184 321A), since neither system is capable of separating the two emission signals.